Fig. 10. Hdm2 F394 inhibits p53-mediated apoptosis more efficiently than wild-type Hdm2. (A) H1299 cells were transfected with expression plasmids for wild-type p53 (3 µg, black bar), either alone or together with increasing amounts (4 µg, dark gray bar; 8 µg light gray bar) of expression plasmids for wild-type Hdm2 or Hdm2 F394. At 55 h post-transfection, floating and adherent cells were collected, fixed and stained for p53 expression. Cells were simultaneously stained for DNA with propidium iodide. Stained cells were subjected to flow cytometric analysis as described previously (Haupt et al., 1996). Cells transfected with p53 were collected and their cell cycle distribution was determined. The sub-G₁ region represents apoptotic subpopulation (Haupt et al., 1996). The results of one representative experiment, in triplicate, are summarized. (B) The same assay as in (A), but in Saos-2 cells. In this experiment, 3 µg of expression plasmid for p53 and 5 µg of expression plasmid for Hdm2 were used. The result of one representative experiment, in triplicate, is shown. The standard error is also given.